ATP-binding cassette (ABC) transporters constitute the largest family of transporters {1-4). It includes both exporters and importers of solutes ranging in size from small molecules to entire protein domains. Eukaryotic ABC transporters predominantly extrude hydrophobic molecules (5), while most bacterial ABC transporters import essential nutrients. The functional unit of an ABC transporter consists of two molecular motors with nucleotide (ATP) binding and hydrolysis domains (NBD or ATP cassette), each coupled to a transmembrane domain (TMD) that encodes the determinants of substrate specificity and provides the binding chamber and passageway across the membrane. The molecular organization of the four domains of ABC transporters was gleaned from crystal structures of a number of ABC importers {6-9) as well as the bacterial multidrug efflux systems Savl866(^0) and MsbA {11). A subclass of ABC exporters has been implicated in multidrug resistance (MDR). Human P-glycoprotein (Pgp) and LmrA from Lactococcus lactis are capable of extruding a large variety of drug molecules;the former providing a strategy for tumor cells to evade the toxicity of chemotherapeutic 6rugs{12-14). This Bridging Project aims to answer a major outstanding question in the field, namely to characterize the nature and amplitude of the conformational motions that transduce the ATP energy input to transport of drugs. Recent crystal structures of bacterial ABC exporters, Sav1866 and MsbA {10, 11), along with extensive spin labeling analysis of MsbA in liposomes {15-18) define a blueprint of the conformational changes induced by ATP binding. However, these investigations were carried out in the absence of drug or substrate. The resulting "minimalist" two-state model is not compatible with biochemical analysis of Pgp that identified at least six intermediates in the transport cycle {19). The missing link is an understanding of the conformational dynamics of ABC transporters as they cycle between transport intermediates. The nature of this problem calls for methods capable of investigating the structure of ABC transporters in their native environment with sufficient spatial resolution and dynamic sensitivity to link structure and function. Pgp provides an ideal system for spectroscopic analysis of functional dynamics. In addition to its direct medical significance, a wealth of information has been accumulated describing its interaction with substrates, including a detailed thermodynamic and kinetic analysis {19, 20). Furthermore, Dr. Al-Shawi has already initiated spin labeling analysis of Pgp {20). The recently determined crystal structure of nucleotide-free {apo) Pgp {21) provides an excellent starting point for experimental design and computational studies of the dynamics, and a context to interpret spectroscopic data. Pgp was captured in an inward-facing conformation where the two symmetry-related halves, each consisting of six helices, are packed in V-shaped geometry, resulting in a cavity open to the cytoplasm and the inner leaflet of the bilayer (Fig. IB). The crystal structure also pinpoints putative drug entry portals near the water/membrane interface that allow access to the cavity. This structure was interpreted mechanistically as a pre-transport state ready to bind drugs. This structure provokes a number of important questions. Very likely apo Pgp needs to sample a large conformational ensemble to accommodate the spectrum of transported substrates. One of these conformations is selectively stabilized by contacts in the crystal lattice. Thus, whether the crystal structure captures the most populated conformer in the membrane needs to be tested. Transported substrates stimulate the ATPase activity and their binding is expected to be signaled to the NBDs through induced conformational changes (22). However, virtually no changes were observed in the substrate-bound crystal structure of Pgp further reinforcing concerns of conformational selectivity {23). In light of these questions. Aim 1 focuses on determining whether the crystalized apo structure reflects the average conformation in the membrane and defines the conformational changes induced by various classes of Pgp substrates. We will also determine the accessibility of the cavity and analyze the environments in the putative entry portals following substrate binding. If indeed the apo state is open to the cytoplasm, ATP binding and hydrolysis are predicted to lead to substantial structural rearrangements. The blueprint of these can be gleaned from the nucleotide bound structures of MsbA and Sav1866 (Fig. 1A). The two NBDs form the canonical ATP sandwich;the TMDs undergo alternating access whereby the cavity closes to the cytoplasm and the inner bilayer leaflet and opens to the extracellular side. Underiying this reconfiguration are large distance changes on the cytoplasmic side and extensive repacking of transmembrane helices. To create the extracellular opening, a twisting motion repacks the TM helices changing the identity of the swapped helices between the two halves of MsbA. We generated a fully energy-minimized homology model of human Pgp in an outward-facing conformation based on the AMPPNP containing structure of MsbA {11) (Fig. 1A). Assuming that the new structure of apo mouse Pgp (ABCB1a)(23) represents Pgp in an inward-facing conformation (Fig. IB), large amplitude conformational changes are predicted between these two key states during multi-drug transport (Fig.lC). Aims 2 and 3 propose to test the MsbA-centric model of ATP-induced conformational change in the presence of the various classes of Pgp substrates. The investigations described below will facilitate a molecular description of the multi-drug efflux phenomenon mediated by Pgp. Structural intermediates inaccessible to other methods of analysis will be defined and tested. The structure and dynamics of key functional intermediates and the nature of conformational changes between them may eventually provide templates for the rational design of specific modulators of Pgp function.